American molecular biologists have made hematopoietic human cells encode non-classical amino acids and incorporate them into proteins. It turned out that the cells retain the ability to differentiate and unable to survive in mice. This can be useful to obtain human proteins with altered properties, for example, to immediately check their pharmacological properties in vivo. Work published in the journal Proceedings of the National Academy of Sciences.
Write the new amino acid in the genetic code is not as difficult as it may seem. The fact that our genetic code is already available “the words” of three nucleotides, which in themselves do not encode. They serve as stop codons. As soon as the ribosome, which builds a protein, it comes to this “meaningless words”, it stops and the protein is finished. And that stop codons can be artificially attributed to a new value.
In order to have a triplet of nucleotides has a value, it is necessary that the cell is able to decode it. This usually happens to the RNA sticks transfer RNA, which carries the desired amino acid. For stop codons, such transport of RNA in a cell no, but you can add it from the outside. And will have to add more enzyme that joins RNA with non-canonical amino acid. Then the cell will incorporate the new amino acid in place of the stop codon.
Similar experiments have been placed in different organisms, from bacteria to mice. And now a group of scientists from the Institute of SCRIPPS under the direction of Peter Schultz (Peter Schultz) got to the human cells. In their paper, the researchers made one of the stop codons (UAG) to encode the amino acid pyrrolidin the same way as do some bacteria and archaea.
To achieve their goal, the authors infected human cells by the viral vector based on Epstein-Barr. The genes of the virus were needed to this DNA segment continued to exist and multiply inside the nucleus. In addition, the vector contained the gene for transfer RNA for pyrrolysine and enzyme transferases that connects pyrrolidin with RNA. Finally, inside the vector was gene green fluorescent protein with the mutation: one of his codons was artificially replaced by the stop codon UAG. This is needed to test the effectiveness of rewriting: if the cell has learned to incorporate pyrrolysine instead of stop codon, then the protein will be full-sized, and glowing. If not learned, then the protein is broken in the middle, and the glow is not visible.
First, scientists tested their method on embryonic cells of human kidney. As expected, they began to glow only when adding pyrrolysine. The researchers then took blood-forming stem cells from umbilical cord blood of man. Successful integration they have achieved about half of the cells, and almost a third of them gained the ability to glow.
These cells then hoisted immunodeficient mice to see how they remain viable and new properties in vivo. It turned out that they’re three times worse than root, than cells that were not injected viral vector (9,5% of cells in the bloodstream and 27.8 percent, respectively). However, when the researchers increased the number of transplanted cells, they have ensured that a quarter of hematopoietic cells in mice proved to be human. About 20 percent of them continued to glow. Then they will make sure that of these human cells to produce different types of formed elements, including different types of white blood cells, and some of them retain the ability to glow.
Thus, the authors were able to get human stem cells to encode non-canonical amino acids. This method can be useful to obtain human proteins with altered properties — the structure or functions — for research or drug development. The authors, for example, suggested that immune cells that originate from these blood cells, can produce modified antibodies, so it would be interesting to test the response of the chimeric mice from their experiment on some allergens.
We have already talked about how molecular biologists rewrote, and then cut the genetic code of Escherichia coli, added to the genetic alphabet of four “letters” and found unconventional code on one of the parasitic plants. Read more about how and why scientists are building and rewriting the genomes of organisms, read our article “With the right to correspondence”.